Early-life environmental cigarette smoke (ECS) exposure alters airway innervation and increases the incidence of asthma later in life, but the mechanisms remain undefined. We recently identified neuronal P-Rex1 as an important regulator of airway innervation and its expression was markedly down-regulated in mice exposed to early-life ECS. Objective: To define the mechanism and importance of P-Rex1 repression in early-life ECS- induced airway hyperinnervation and hyperresponsiveness (AHR), the pathophysiologic hallmark of asthma. Long-term goal: to determine whether targeting neuronal P-Rex1 provides a new strategy for preventing early-life ECS-related asthma progression. Findings: 1) P-Rex1 is highly expressed in neurons but not airway cells. 2) P-Rex1 knockout (KO) mice exhibit airway smooth muscle (ASM) hyperinnervation and AHR. WT mice exposed to early-life ECS showed similar phenotypes with 60% reduction of P-Rex1 in vagal ganglia. Severing vagus nerves attenuated AHR of these mice. 3) ECS exposure enhances brain-derived neurotrophic factor (BDNF) secretion from ASM cells, serving as a target-derived signal for neurite growth of mouse vagal sensory neurons in vitro. 4) P-Rex1 over-expression blocked BDNF-stimulated neurite growth whereas loss of P-Rex1 markedly sensitized these neurons to BDNF stimulation. 5) ECS-elevated interleukin (IL)-6 down-regulates P- Rex1 and enhances BDNF-stimulated neurite growth that is blocked by a PKC inhibitor. Hypothesis: IL-6 repression of neuronal P-Rex1 plays a crucial role in early-life ECS-induced ASM hyperinnervation and AHR of asthma. We will test this hypothesis using molecular, cellular, and animal models. Aim 1: To elucidate the mechanism of early-life ECS-exposure-induced neuronal P-Rex1 repression. We hypothesize that IL-6 represses neuronal P-Rex1 via a PKC-dependent mechanism. We will first use siRNAs to silence P-Rex1 in mouse vagal sensory neurons to assess the importance of P-Rex1 in IL-6 potentiation of BDNF-induced neurite growth. We will then investigate if restoration of P-Rex1 expression attenuates IL-6 stimulatory effects. Finally, we will use inhibitors and siRNAs to identify the PKC isoforms responsible for IL-6-induced P-Rex1 repression and neurite growth. Aim 2: To investigate the pathologic importance of neuronal P-Rex1 repression in early-life ECS exposure-related asthma. We hypothesize that IL-6 repression of neuronal P-Rex1 is a critical determinant in the development and severity of early-life ECS-related asthma. WT and P-Rex1 KO mice will be exposed to ECS or air for 10 days beginning on postnatal day (PND) 2. AHR will be assessed by invasive tracheostomy 24h after a re-exposure of mice to acute insult of ECS or allergen house dust mite on PND59. Effects of early-life ECS exposure on ASM innervation and phenotype (remodeling, contractility) will be examined. Whether loss of P-Rex1 exacerbates early-life ECS-induced ASM hyper-innervation and AHR will be determined. Finally, we will determine whether oral administration of IL-6 inhibitor LMT-28 ameliorates early-life ECS-induced mouse AHR by preventing P-Rex1 repression and ASM hyperinnervation.